Formation of electronically excited states during the interaction of p-benzoquinone with hydrogen peroxide

The reaction between p-benzoquinone and H₂O₂ in slightly alkaline solutions yields three major quinoid products that accumulate in the reaction mixture: (a) 2,3-epoxy-p-benzoquinone, (b) 2-hydroxy-p-benzoquinone and (c) p-benzohydroquinone. The reaction is accompanied by photoemission, probably originating from excited triplet 2-hydroxy-p-benzoquinone. These products originate from hydrogen peroxide and hydroxide nucleophilic addition to the C₂?C₃ double bond, as well as secondary redox interactions. The hydroxy substituent and the epoxide ring exert a substantial influence on the electronic distribution in the p-benzoquinone molecule leading to a decrease in the half-wave potential, as compared to the parent p-benzoquinone. The generation of electronically excited states is the result of reactions secondary to the nucleophilic additions involving 2-hydroxy-p-benzosemiquinone, H₂O₂ and hydroxyl radical. The process involves the primary oxidation of 2-hydroxy-p-benzosemiquinone by hydrogen peroxide, followed by oxidation of the semiquinone by hydroxyl radical leading to the formation of the electronically excited quinone. The decay of the excited triplet to the ground state is accompanied by photoemission with maximal intensity at 485–530 nm. Thermodynamic calculations along with an observed increase of photoemission intensity in anaerobiosis point to the triplet (n, π*) multiplicity of the excited state. The efficiency of chemiluminescence could be calculated as 10^?8 photons/2-hydroxy-p-benzoquinone molecule formed. Photoemission arising from the p-benzoquinone/H₂O₂ reaction was inhibited efficiently by addition of GSH to the reaction mixture. This may be due to deactivation of the triplet quinone by a 2-glutathionyl-p-benzohydroquinone adduct, involving thioether α-hydrogen atom-transfer to the triplet ketone.     

In vivo and in vitro oxidative biotransformation of dimethylformamide in rat

In rats and in humans, dimethylformamide (DMF) is mainly metabolized into N-hydroxymethyl-N-methylformamide (DMF-OH). The in vitro oxidation of DMF by rat liver microsomes is decreased in the presence of catalase and superoxide dismutase. The radical scavengers, dimethylsulfoxide (DMSO), tertiary butyl alcohol (t-butanol), aminopyrine, hydroquinone and trichloroacetonitrile reduce the oxidation of DMF to DMF-OH in vitro and in vivo. Conversely, DMF inhibits the demethylation of DMSO, t-butanol and aminopyrine. The addition of iron-EDTA to the incubation system induces the production of N-methylformamide (NMF) from DMF. These results support the hypothesis that the metabolic pathway leading from DMF to DMF-OH and NMF involves hydroxyl radicals. Superoxide radical and hydrogen peroxide take part in the metabolic process. DMF is preferentially metabolized into DMF-OH. NMF appears mainly when the production of hydroxyl radicals is stimulated, the methyl group being recovered as formic acid.     

Detection and identification of sulfhydryl conjugates of p-benzoquinone in microsomal incubations of benzene and phenol

The glutathione and cysteine conjugates of p-benzoquinone are detected and conclusively identified in microsomal incubations of benzene and phenol using liquid chromatography/electrochemistry (LCEC). Identification of the compounds is based on retention time, electrochemical behavior and acid hydrolysis. The fact that both of these compounds can be detected easily in a benzene incubation provides further evidence that p-benzoquinone or the corresponding semiquinone is a product of benzene metabolism in vivo. The conjugation of p-benzoquinone with glutathione is predominantly a nonenzymatic process. This is illustrated by the fact that the addition of cytosolic glutathione-S-transferases do not significantly increase the amount of glutathione conjugate produced in a phenol incubation containing glutathione.The kinetic constants for phenol metabolism to hydroquinone by microsomal protein are calculated. As suspected, the rate of metabolism of phenol is significantly higher than the rate of benzene metabolism. The V_max for phenol metabolism was calculated to be 7.1 nmol/min/mg protein and the K_M was found to be 0.38 mM.The further oxidation of hydroquinone to p-benzoquinone appears to be primarily an enzymatic process. Incubations of just hydroquinone with glutathione at 37°C produced only a small amount of the glutathione conjugate. The addition of cytosolic protein increases the amount of p-benzoquinone produced about 10-fold. This could be due to the peroxidases found in that medium. The addition of microsomal protein and NADPH increases the amount of glutathione conjugate produced to over 100-fold of that produced nonenzymatically. This indicates that a microsomal enzyme is responsible for the oxidation of hydroquinone to p-benzoquinone in vitro and the subsequent covalent binding to macromolecules.     

Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp

A Moraxella strain grew on p-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on p-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2′-dipyridyl, p-nitrophenol was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of p-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to γ-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD⁺, γ-hydroxymuconic semialdehyde was converted to β-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NAD were required and γ-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from p-nitrophenol-growth cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of p-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone. 1,4-Benzoquinone, a likely intermediate in the initial reaction, was not detected. Hydroquinone is converted to β-ketoadipic acid via γ-hydroxymuconic semialdehyde and maleylacetic acid.     

Flavin-containing monooxygenases from <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> responsible for fungal metabolism of phenolic compounds

We investigated the cellular responses of the white-rot basidiomycete Phanerochaete chrysosporium against vanillin. Based upon a proteomic survey, it was demonstrated that two flavin-containing monooxygenases (PcFMO1 and PcFMO2) are translationally up-regulated in response to exogenous addition of vanillin. To elucidate their catalytic functions, we cloned cDNAs and heterologously expressed them in Escherichia coli. The recombinant PcFMO1 showed catalytic activities against monocyclic phenols such as phenol, hydroquinone, and 4-chlorophenol. In addition, the product from hydroquinone was identified as 1,2,4-trihydroxybenzene, an important intermediate in a metabolic pathway of aromatic compounds in which the aromatic ring of 1,2,4-trihydroxybenzene can be further cleaved by fungal dioxygenases for mineralization. Thus, the ortho-cleavage pathway of phenolic compounds would presumably be associated with PcFMO1.     

Multi-step metabolic activation of benzene. Effect of superoxide dismutase on covalent binding to microsomal macromolecules,and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry

Incubation of [¹⁴C]benzene or [¹⁴C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accumulation of [¹⁴C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p-benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectrometry (FDMS) as 2-(S-glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p-benzoquinone in the presence of [³H]glutathione or, alternatively, with [¹⁴C]benzene or [¹⁴C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-(S-glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [³H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p-benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-(S-glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH⁺) (m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities.The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p-benzosemiquinone and/or p-benzoquinone as quantitatively important reactive metabolites.     

Decomposition Pathways of p-Bromophenol on γ-Irradiation in Aqueous Systems

In order to elucidate the radiolysis mechanism of p-bromophenol, quantitative determination of the radiolysis products was carried out by gas chromatography and polarography. G(?p · BP) and G(Br^?) were 3.86 and 2.58 at neutral pH, and 1.09 and 0.26 at pH 1.0, respectively, This, together with the radical scavenger effects indicated that hydrated electrons contribute principally to the degradation of p-bromophenol through debromination, followed by the formation of dimer and trimer products by phenylation of the resulting p-hydroxyphenyl radical. This chain-like reaction may cause the difference (G-value = 1.28) between G(?p· BP) and G(Br^?). The contribution of OH radicals to G(?p· BP) is known to be small as compared with other aromatic compounds, because of the poor yield of hydroxylated products such as hydroquinone, 4-bromocatechol and 4-bromoresorcinol.     

E.S.R. study of alkaline,oxidative degradation of saccharides. Identification of 2,5-dihydroxy-p-benzosemiquinone

The formation of radical intermediates during alkaline, oxidative degradation of saccharides and α-hydroxycarbonyl compounds has been studied by e.s.r. spectroscopy. Quantum chemical calculation and experiments in alkaline D₂O solution showed that the dominant component of the overall spectrum corresponds to 2,5-dihydro-p-benzosemiquinone. Formation of this radical was also observed in the alkaline-degradation products of cellulose, starch, and (4-O-methylglucurono)xylan in the presence of air.     

Reaction of imidazole and hydroquinone with oxymyoglobin

Oxymyoglobin reacts with imidazole, substituted imidazoles, and hydroquinone to give metmyoglobin. The kinetics of these reactions have been studied. The rates are first order in both reactants, and second-order rate constants are reported. At pH 8.2, k₁ for imidazole is 2.5 ± 0.3 × 10^?3 M^?1 sec^?1 and for hydroquinone is 4 ± 0.4 × 10^?1 M^?1 sec^?1. The rates are independent of pH for imidazole but increase rapidly with pH for hydroquinone. The mechanism for all these reactions is thought to involve the two-electron reduction of molecular oxygen to peroxide with concurrent oxidation of both the protein and the reactant. An analogous mechanism has been suggested previously [1] for the reaction of oxyhemoglobin with hydroquinone. It has previously been shown [6] that imidazole can mediate the transfer of electrons to heme proteins by forming a transient reduced radical. The present results indicate that it can also form a transient oxidized radical under mild conditions. This dual capability may be important in biological electron-transfer processes.     

Study of aldehyde oxidase-catalyzed metabolic pathway of phenanthridine using MCR-ALS method

Evaluation of metabolic pathways is one of the challenging areas in biological and pharmaceutical sciences. Phenanthridine oxidation to phenanthridinone is used commonly to study aldehyde oxidase activity. This reaction could pass through phenanthridine N-oxide intermediate. In the present study, the application of multivariate curve resolution, optimized by alternating least squares (MCR-ALS) to investigate this metabolic pathway has been described. The results obtained from MCR-ALS analysis along with those obtained from the use of potassium ferrocyanide method indicated that phenanthridine is directly oxidized to phenanthridinone by rat liver aldehyde oxidase without passing through phenanthridine N-oxide intermediate. It was also found that the later compound is not metabolized by this enzyme.     

Rat liver microsomal enzyme catalyzed oxidation of 4-phenyl-trans-1-(2-phenylcyclopropyl)-1,2,3,6-tetrahydropyridine.

As part of our ongoing studies to characterize the catalytic pathway(s) for the monoamine oxidase and cytochrome P450 catalyzed oxidations of 1,4-disubstituted 1,2,3,6-tetrahydropyridinyl derivatives, we have examined the metabolic fate of 4-phenyl-trans-1-(2-phenylcyclopropyl)-1,2,3,6-tetrahydropyridine in NADPH supplemented rat liver microsomes. Three metabolic pathways have been identified: (1) allylic ring alpha-carbon oxidation to yield the dihydropyridinium species, (2) nitrogen oxidation to yield the N-oxide and (3) N-dealkylation to yield 4-phenyl-1,2,3,6-tetrahydropyridine and cinnamaldehyde. A possible mechanism to account for the formation of cinnamaldehye involves an initial single electron transfer from the nitrogen lone pair to the iron oxo system Fe(+3)(O) to form the corresponding cyclopropylaminyl radical cation that will be processed further to the final products. The reaction pathway leading to the dihydropyridinium metabolite may also proceed via the same radical cation intermediate but direct experimental evidence to this effect remains to be obtained.     

Identification by electron spin resonance of free radicals formed during the oxidation of 4-hydroxyanisole catalyzed by tyrosinase

We have observed the formation of free radicals during the oxidation of the melanocytotoxic agent 4-hydroxyanisole with the enzyme tyrosinase as a catalyst. The first free radical to form is identified as the 4-methoxy-1,2-benzosemiquinone radical anion. The peak concentration of this radical increases with tyrosinase concentration; a minimum concentration of 50 micrograms/ml of tyrosinase was needed to observe this radical. The peak concentration of this radical is independent of 4-hydroxyanisole concentration. This radical is produced by reverse dismutation of the primary product, 4-methoxy-1,2-benzoquinone and 4-methoxycatechol produced indirectly.     

Microbial manganese(III) reduction fuelled by anaerobic acetate oxidation

Soluble manganese in the intermediate +III oxidation state (Mn³⁺) is a newly identified oxidant in anoxic environments, whereas acetate is a naturally abundant substrate that fuels microbial activity. Microbial populations coupling anaerobic acetate oxidation to Mn³⁺ reduction, however, have yet to be identified. We isolated a Shewanella strain capable of oxidizing acetate anaerobically with Mn³⁺ as the electron acceptor, and confirmed this phenotype in other strains. This metabolic connection between acetate and soluble Mn³⁺ represents a new biogeochemical link between carbon and manganese cycles. Genomic analyses uncovered four distinct genes that allow for pathway variations in the complete dehydrogenase‐driven TCA cycle that could support anaerobic acetate oxidation coupled to metal reduction in Shewanella and other Gammaproteobacteria. An oxygen‐tolerant TCA cycle supporting anaerobic manganese reduction is thus a new connection in the manganese‐driven carbon cycle, and a new variable for models that use manganese as a proxy to infer oxygenation events on early Earth.     

Plasticity of a key metabolic pathway in fungi

Beta oxidation is the principal metabolic pathway for fatty acid degradation. The pathway is virtually universally present throughout eukaryotes yet displays different forms in enzyme architecture, substrate specificity, and subcellular location. In this review, we examine beta oxidation across the fungal kingdom by conducting a large-scale in silico screen and localization prediction for all relevant enzymes in >50 species. The survey reveals that fungi exhibit an astounding diversity of beta oxidation pathways and shows that the combined presence of distinct mitochondrial and peroxisomal pathways is the prevailing and likely ancestral type of beta oxidation in fungi. In addition, the available information indicates that the mitochondrial pathway was lost in the common ancestor of Saccharomycetes. Finally, we infer the existence of a hybrid peroxisomal pathway in several Sordariomycetes, including Neurospora crassa. In these cases, a typically mitochondrion-located enzyme compensates for the lack of a peroxisomal one. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microbial metabolism of alkylbenzene sulphonates Fungal metabolism of 1-phenylundecane-p-sulphonate and 1-phenyldodecane-p-sulphonate

A study was made of the biodegradation of 1-phenylundecane-p-sulphonate and 1-phenyldodecane-p-sulphonate byCladosporium resinae (CMI 88968) which was capable of growth on a number of such alkylbenzene sulphonate homologues as the sole source of carbon and sulphur. The results from both whole-cell and cell-free systems indicated that the alkyl, aryl and sulphonate moieties of detergent homologues were metabolised by the fungus. The alkyl side-chain, after a presumed initial oxidation of the terminal methyl group, was subsequently oxidised by a β-oxidation pathway. Three enzymes of the β-oxidation pathway, i.e. acyl-CoA synthetase, acyl-CoA dehydrogenase and β-hydroxyacyl-CoA dehydrogenase were identified in detergent-grown cell-free extracts of the fungus. The sulphonate moiety was released as sulphate by a desulphonating enzyme. The combined results of continuous sampling programmes monitored by both TLC and sulphate appearance in the growth medium indicated that desulphonation of the aromatic moiety was an early event in the overall biodegradation of synthetic detergent homologues. The presence of 1-phenylvalerate, 1-phenylpropionate, benzoate,p-hydroxybenzoate and 3,4-dihydroxybenzoate in cells after growth on 1-phenylundecane-p-sulphonate was indicated by GLC analysis. Cells grown on 1-phenyldodecane-p-sulphonate were shown to contain 1-phenylhexanoate, 1-phenylbutyrate, phenylacetate,p-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate. The aromatic nuclei remaining after alkyl side-chain biodegradation were further metabolised by an oxidation sequence involving an “ortho-cleavage” pathway. An overall metabolic pathway for the biodegradation of alkylbenzene sulphonates byCladosporium resinae is proposed.     

Structural characterization of 2,6‐dichloro‐p‐hydroquinone 1,2‐dioxygenase (PcpA) from Sphingobium chlorophenolicum,a new type of aromatic ring‐cleavage enzyme

PcpA (2,6‐dichloro‐p‐hydroquinone 1,2‐dioxygenase) from Sphingobium chlorophenolicum, a non‐haem Fe(II) dioxygenase capable of cleaving the aromatic ring of p‐hydroquinone and its substituted variants, is a member of the recently discovered p‐hydroquinone 1,2‐dioxygenases. Here we report the 2.6 Å structure of PcpA, which consists of four βαβββ motifs, a hallmark of the vicinal oxygen chelate superfamily. The secondary co‐ordination sphere of the Fe(II) centre forms an extensive hydrogen‐bonding network with three solvent exposed residues, linking the catalytic Fe(II) to solvent. A tight hydrophobic pocket provides p‐hydroquinones access to the Fe(II) centre. The p‐hydroxyl group is essential for the substrate‐binding, thus phenols and catechols, lacking a p‐hydroxyl group, do not bind to PcpA. Site‐directed mutagenesis and kinetic analysis confirm the critical catalytic role played by the highly conserved His10, Thr13, His226 and Arg259. Based on these results, we propose a general reaction mechanism for p‐hydroquinone 1,2‐dioxygenases.     

Thiodiglycol Metabolism in Alcaligenes xylosoxydans subsp. denitrificans

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C–S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO₄ ^2– ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.     

Characterization of a glutathione conjugate of the 1,4-benzosemiquinone-free radical formed in rat hepatocytes

Rat hepatocytes treated with 1,4-benzoquinone formed 1,4-benzosemiquinone and 2-S-glutathionyl-1,4-benzosemiquinone radicals as detected by ESR spectroscopy. The 2-S-glutathionyl-1,4-benzosemiquinone radical was first obtained from the reaction of 1,4-benzoquinone with glutathione. Glutathione both reduced benzoquinone to form benzosemiquinone and conjugated benzoquinone to form 2-S-glutathionyl-1,4-benzosemiquinone radical. The ratio of these two radicals depended upon the ratio of 1,4-benzoquinone to glutathione. At near equimolar ratios, the 2-S-glutathionyl-1,4-benzosemiquinone radical was predominantly formed. This radical was characterized by computer simulation of the experimental spectra and identified by comparison of its hyperfine coupling constants with those of chemical analogues. The 2-S-glutathionyl-1,4-benzosemiquinone radicals formed inside hepatocytes, and then crossed the plasma membrane into the media.     

Observations on the bacterial oxidation of chlorophenoxyacetic acids

Summary Three bacterial strains, one ofF. peregrinum (Stapp and Spicher) and two Achromobacter strains, have been isolated from soil and shown to decompose either 2,4-D, MCPA orp-chlorophenoxyacetic acid. Aerobic conditions are essential for the bacterial decomposition of 2,4-D. Pretreatment of soil with one of the three chlorophenoxyacetic acids accelerated the rate of breakdown of either of the other two. In a liquid medium, growth of theF. peregrinum strain caused breakdown of 2,4-D and liberated 76% of the chlorine in 2,4-D in ionic form. An unknown acidic substance, colourless in acid solution but forming a yellow sodium salt has been detected in cultures ofF. peregrinum or an MCPA-decomposing Achromobacter strain growing inp-chlorophenoxyacetate medium. The bacterial oxidation of chlorophenoxyacetic acid herbicides was attributed to adaptive enzyme formation. Respiration experiments showed that the oxidation of 2,4-D or ofp-chlorophenoxyacetic acid is incomplete. 4-Chloro-2-hydroxyphenoxyacetic acid and 4-chlorocatechol may be metabolic intermediates in the case ofp-chlorophenoxyacetic acid, but no intermediary metabolites have as yet been established for 2,4-D.     

Peroxidase catalysed oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone

Horseradish peroxidase catalysed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone and subsequent oxidation of the hydroquinone to methoxy-p-benzoquinone. Peroxidase also catalysed the oxidation of vanillyl alcohol to vanillin and vanillic acid; however, neither vanillyl alcohol nor vanillin appeared to give rise to methoxyhydroquinone directly. Correspondingly, peroxidase catalysed the oxidative decarboxylation of syringic acid to 2,6-dimethoxy-p-hydroquinone and subsequent oxidation of the hydroquinone to 2,6-dimethoxy-p-benzoquinone.